Biomedical subjects

Masaki Takiguchi

Publications and source records attributed to Masaki Takiguchi.

28 records · Page 2Linked to original sources

PubMed2003

Drug-sensitivity pattern analysis for study of functional relationship between gene products.

We have developed a method that we call 'drug-sensitivity pattern analysis', or DSPA, for analysis of protein function. Cells are transfected with cDNA of the test molecule, followed by analysis of the sensitivity of the transfected cells to multiple growth-inhibitory drugs. If two cDNA products have similar functions, their transfected cells should show similar drug-sensitivity patterns. The cDNAs of some signaling molecules were transfected into NIH3T3 or Ha-ras-transformed NIH3T3 (ras-NIH) cells and stable transfectants, which expressed high amounts of the gene product, were isolated. Chemosensitivity of the transfected clone was compared with the parental cells by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method using more than 40 drugs. The chemosensitivity changes caused by the transfected gene were calculated and expressed numerically as 'drug chemosensitivity index' (DCI). When the DCI values were analyzed by regression analysis, a significant positive relationship between IkappaBalpha superrepressor and dominant-negative IKKbeta and an inverse relationship between p53 and Mdm2 were consistent with previous reports. Thus, the DSPA method is useful for identifying functional similarities between gene products.

3T3 Cells↗

PubMed2003

Gene expression profiling reveals the mechanism and pathophysiology of mouse liver regeneration.

Comprehensive analysis of the changes in gene expression during liver regeneration was carried out by using an in-house microarray composed of 2,304 distinct mouse liver cDNA clones. Mice were subjected to partial two-thirds hepatectomy, and changes in mRNA levels were monitored up to 48 h. Of the 2,304 genes analyzed, 496 genes showed expression levels measurable at all time points after the partial hepatectomy. 317 genes were up- or down-regulated 2-fold or more at least at one time point during liver regeneration and were classified into eight clusters based on their expression patterns. With a more stringent cut-off value of +/-2 S.D., 68 genes were listed and were classified into five clusters. In these two analyses with different clustering criteria, functionally categorized genes showed similar cluster distributions. Genes involved in protein synthesis and posttranslational processing were significantly enriched in the cluster characterized by rapid gene activation and subsequent persistence. This suggests the importance of modulating the efficiency of protein supply and/or altering the composition of protein population from the early phase of hepatocyte proliferation. Genes for two major liver functions, i.e. plasma protein secretion and intermediate metabolism were enriched in distinct clusters exhibiting the features of gradual gene activation and sustained repression, respectively. Therefore, these genes are differentially regulated during the regeneration, possibly leading to changes in the flow of amino acids and energy from enzyme proteins to plasma proteins in their synthesis. Thus, clustering analysis of expression patterns of functionally classified genes gave insights into mechanism and pathophysiology of liver regeneration.

Animals↗

PubMed2003

Two-peaked synchronization in day/night expression rhythms of the fibrinogen gene cluster in the mouse liver.

Genes expressed with day/night rhythms in the mouse liver were searched for by microarray analysis using an in-house array harboring mouse liver cDNAs. The rhythmic expression with a single peak and trough level was confirmed by RNA blot analysis for 3beta-Hsd and Gabarapl1 genes exhibiting a peak in the light phase and Spot14, Hspa8, Hspa5, and Hsp84-1 genes showing a peak in the dark phase. On the other hand, mRNA levels for all of the three fibrinogen subunits, Aalpha, Bbeta and gamma, exhibited two peaks each in the light and dark phases in a synchronized manner. This two-peaked rhythmic pattern of fibrinogen genes as well as the single peak-trough pattern of other genes was diminished or almost completely lost in the liver of Clock mutant mice, suggesting that the two-peaked expression is also under the control of oscillation-generating genes. In constant darkness, the first peak of the expression rhythm of fibrinogen genes was almost intact, but the second peak disappeared. Therefore, although the first peak in the subjective day is a component of the innate circadian rhythm, the second peak seems to require light stimuli. Fasting in constant darkness caused shifts of time phases of the circadian rhythms. Protein levels of the fibrinogen subunits in whole blood also exhibited circadian rhythms. In the mouse and human loci of the fibrinogen gene cluster, a number of sequence elements resembling circadian transcription factor-binding sites were found. The fibrinogen gene locus provides a unique system for the study of two-peaked day/night rhythms of gene expression in a synchronized form.

Animals↗

PubMed2003

Comparing gene expression profiles in human liver, gastric, and pancreatic tissues using full-length-enriched cDNA libraries.

In the post-genome-sequencing era, full-length cDNA-sequence resources are extremely useful for functional analyses of genes. In addition, comprehensive gene profiling of human tissues at the mRNA level is also useful in understanding the molecular mechanisms of tissue-specific functions and disease pathogenesis. In this study, to obtain a wide variety of full-length cDNA clones derived from digestive tissues, numerous expressed sequence tags were generated from libraries enriched with full-length cDNAs. In total, 13575 sequences were obtained from three cDNA libraries, which were constructed from tissues and cell lines of human liver, stomach, and pancreas. The integration of overlapping clones categorized the sequences into 5936 clusters (1666, 2746, and 2222 clusters in the liver, stomach, and pancreas, respectively). Of these, 1138 clones were scored as full-length cDNAs. Surprisingly, the redundant clones from all three tissues were assembled to show that only 101 genes (1.7% of the assembled 5936 genes) were shared. These results suggest that functional differences between tissues are probably related to their divergent gene expression profiles, and form a basis for understanding the molecular mechanisms underlying tissue-specific pathogenesis that are expressed in different organs. In addition, the full-length cDNAs obtained in this study should prove useful for future functional analyses of the genes expressed in digestive tissues.

Journal Article↗

PubMed2003

Acquisition of drug resistance by constitutive suppression of NF-kappaB in ras-transformed NIH3T3 mouse fibroblasts.

Apoptotic cell death is frequently suppressed by NF-kappaB transcription factor. We examined the effects of NF-kappaB on the sensitivity to anti-cancer drugs by using two NF-kappaB-inhibitory molecules, IkappaBalpha-super-repressor (IkappaBalpha-SR) and dominant negative IKKbeta (IKKbeta-DN). Ha-ras-transformed NIH3T3 (ras-NIH3T3) mouse fibroblasts were stably transfected with these cDNAs, and suppression of NF-kappaB activity was confirmed by electrophoretic mobility shift assays. Cell viability analysis revealed that IkappaBalpha-SR- or IKKbeta-DN-transfected cells were more resistant to peplomycin, mitomycin C, and camptothecin. Flow cytometric analysis indicated partial G1 arrest of these transfected cells. Consistently, elevated expression of IkappaBalpha-SR and IKKbeta-DN was accompanied by increased levels of p21 but not of Bax. Transfection of p21 into ras-NIH3T3 cells caused similar reduced chemosensitivity to the anti-cancer drugs. These results collectively indicate that constitutive suppression of NF-kappaB activity reduced the chemosensitivity to anti-cancer drugs via enhanced expression of p21.

Animals↗

PubMed2003

In-house cDNA microarray analysis of gene expression profiles involved in SCC cell lines.

To analyze gene expression in oral cancer, we produced a specialized in-house cDNA microarray. The cDNA library was constructed from surgical specimens of oral squamous cell carcinoma (SCC) using an oligo-capping method. cDNA clones (n=4,800) were randomly selected and their 5'-end nucleotide sequences were determined. Overlapping clones were excluded, and 1,423 independent clones were selected and used for microarray production. Compared to the public nucleotide sequence database, 61% of our cDNA clones were full-length. By correlating expression patterns across SCC cell lines, we identified 53 genes (7 up-regulated and 46 down-regulated) that are differentially expressed in SCC cell lines compared to normal mucosa. Semi-quantitative RT-PCR analysis confirmed these findings and validity of our cDNA microarray. Using specimens from SCC patients, we investigated the expression status of the IL-1ra gene, which showed the down-regulated gene by microarray analysis. Gene expression clearly fell in the SCC specimens relative to their references, which indicated that our in-house cDNA microarray system rapidly identified and characterized candidate biomarkers for clinical use.

Carcinoma, Squamous Cell↗

PubMed2002

Facilitation of adenoviral wild-type p53-induced apoptotic cell death by overexpression of p33(ING1) in T.Tn human esophageal carcinoma cells.

To investigate the effect of p33(ING1) on wild-type p53 gene therapy, T.Tn human esophageal carcinoma cells were stably transfected with p33(ING1) cDNA. Infection with Ad-p53 (recombinant adenovirus containing wild-type p53) into p33-transfected cells reduced cell viability, while infection with empty vector had little effect. This reduced viability was shown to be due to apoptotic cell death by the TUNEL (terminal deoxynucleotidyl transferase-mediated nick end-labeling) assay. Following infection with Ad-p53, levels of p53 were similar in p33-expressing cells and in the parental line. However, levels of p21 and Mdm2 were elevated in p33-transfected cells. Nonetheless, this enhanced expression of Mdm2 appeared to be ineffective in downregulating p53. Transient transfection with mutant Mdm2 prior to Ad-p53 infection provided a significant protection as compared with cells transfected with wild-type Mdm2. These results imply a synergistic effect between p33 and p53 in the induction of apoptosis of human esophageal carcinoma cells. A role for Mdm2 in this synergism is suggested.

Adenoviridae↗

PubMed2002

Regulation of transformed state by calpastatin via PKCepsilon in NIH3T3 mouse fibroblasts.

Ca(2+)-activated neutral protease calpain is ubiquitously expressed and may have pleiotropic biological functions. We have previously reported that repeated treatment of NIH3T3 mouse fibroblasts with the calpain inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) resulted in the induction of transformed foci [T. Hiwasa, T. Sawada, and S. Sakiyama (1990) Carcinogenesis 11, 75-80]. To elucidate further the effects of calpain in malignant transformation of NIH3T3 cells, calpastatin, an endogenous specific inhibitor of calpain, was expressed in NIH3T3 cells by transfection with cDNA. G418-selected calpastatin-expressing clones showed a significant increase in the anchorage-independent growth ability. A similar increase in cloning efficiency in soft agar medium was also observed in calpain small-subunit-transfected clones. On the other hand, reduced expression of calpastatin achieved by transfection with calpastatin antisense cDNA in Ha-ras-transformed NIH3T3 (ras-NIH) cells caused morphological reversion as well as a decrease in anchorage-independent growth. When NIH3T3 cells were treated with ALLN for 3 days, cell growth was stimulated by approximately 10%. This growth stimulation by ALLN was not observed in ras-NIH cells, but recovered by expression of a dominant negative form of protein kinase C (PKC)epsilon but not by that of PKCalpha. Western blotting analysis showed that an increase in PKCepsilon was much more prominent than that of PKCalpha in NIH3T3 cells after treatment with ALLN. These results are concordant with the notion that calpain suppresses malignant transformation by predominant degradation of PKCepsilon.

3T3 Cells↗

PubMed2002

Expression and regulation of the gene for arginase I in mouse salivary glands: requirement of CCAAT/enhancer-binding protein alpha for the expression in the parotid gland.

Arginase in salivary glands is potentially involved in the synthesis of proline, glutamate, and polyamines that play specific physiological roles in the glands, and also in depletion of arginine in the oral cavity to protect teeth from microorganisms. We detected protein and mRNA for the type I isoform of arginase in mouse salivary glands. Enzymes of the arginine-biosynthetic pathway were also detected. Immunohistochemical analysis revealed that arginase I was enriched in the striated duct, and was also present in the acinus, demilune and granulated duct. Mice with targeted disruption of the gene for C/EBPalpha, which is a transcription factor essential for expression of the arginase I gene in the liver, showed dramatically reduced immunoreactivity for arginase I in the parotid gland but not in the submandibular and sublingual glands. Therefore, C/EBPalpha is specifically required for expression of the arginase I gene in the parotid gland.

Animals↗

PubMed2002

Decrease in growth factor receptors after treatment with serine protease inhibitor ONO-3403.

FOY-305 is a synthetic serine protease inhibitor and ONO-3403 is a derivative with a higher protease-inhibitory activity. The growth-suppressive effects of ONO-3403 were more prominent in Ha-ras-transformed NIH3T3 (ras-NIH) cells than in non-transformed NIH3T3 cells. After treatment of ras-NIH cells with ONO-3403 at 100-200 microg/ml, the percentage of cells found in G(1) phase decreased and, concomitantly, that in S phase increased. Molecular events caused by ONO-3403 were investigated by Western blot analysis using anti-phosphotyrosine antibody. The results showed a marked decrease in the tyrosine phosphorylation level of a 180-kDa protein after treatment with ONO-3403. This 180-kDa phosphotyrosine-containing molecule which was tentatively designated pY-p180 might be platelet-derived growth factor (PDGF) receptor since addition of PDGF to serum-starved NIH3T3 cells induced a marked tyrosine phosphorylation of the same size within 5 min. This was further confirmed by immunoprecipitation of cell extract with anti-PDGF-receptor antibody followed by Western blot analysis using anti-phosphotyrosine antibody. Treatment of T.Tn human esophageal carcinoma cells with ONO-3403 caused also decrease in pY-p180, which appeared to be epidermal growth factor receptor. Thus, ONO-3403 may induce growth suppression by down-regulation of cell surface growth factor receptors.

3T3 Cells↗